Purpose: To explore the effect of pulsatile flow in the decellularization process of small blood vessels. Methods: A total of 30 human umbilical cords were used in the current study. The umbilical cords were flushed with 0.25% trypsin/0.01% EDTA for 30 minutes, followed by treatment with 1% sodium dodecyl sulfate sodium dodecyl sulfate for 3 hours. The effectiveness of decellularization on the umbilical artery wall was analyzed by mechanical analysis. The scaffolds' biocompatibility was observed by co-culture with the human umbilical endothelial cells. Results: The maximum stress of the arteries before and after denuding was 3.55 +/- 0.42N and 3.50 +/- 0.43N, respectively. Under the pressure of 300 mmHg, 28 pieces of umbilical cords remained intact before and after the flushing, while 2 pieces ruptured under 300 mmHg. There was no significant difference in mechanical properties between flushed and control arteries. Isolated human umbilical endothelial cells grow and spread well on the decellularized umbilical artery scaffolds. Conclusions: Decellularization by pulsatile flow in human umbilical artery is a convenient, rapid and efficient approach to increase the availability of small caliber blood vessel scaffolds.